title: Identification of breast cancer DNA methylation markers optimized for fine-needle aspiration samples.
abstract: BACKGROUND
  Random periareolar fine-needle aspiration (RP-FNA) is increasingly used in trials of breast cancer prevention for biomarker assessments. DNA methylation markers may have value as surrogate endpoint biomarkers, but this requires identification of biologically relevant markers suitable for paucicellular, lymphocyte-contaminated clinical samples.
  
  
  METHODS
  Unbiased whole-genome 5-aza-2'-deoxycytidine (5AZA)-induced gene expression assays, followed by several phases of qualitative and quantitative methylation-specific PCR (MSP) testing, were used to identify novel breast cancer DNA methylation markers optimized for clinical FNA samples.
  
  
  RESULTS
  The initial 5AZA experiment identified 453 genes whose expression was potentially regulated by promoter region methylation. Informatics filters excluded 273 genes unlikely to yield useful DNA methylation markers. MSP assays were designed for 271 of the remaining genes and, ultimately, 33 genes were identified that were differentially methylated in clinical breast cancer samples, as compared with benign RP-FNA samples, and never methylated in lymphocytes. A subset of these markers was validated by quantitative multiplex MSP in extended clinical sample sets. Using a novel permutation method for analysis of quantitative methylation data, PSAT1, GNE, CPNE8, and CXCL14 were found to correlate strongly with specific clinical and pathologic features of breast cancer. In general, our approach identified markers methylated in a smaller subpopulation of tumor cells than those identified in published methylation array studies.
  
  
  CONCLUSIONS
  Clinically relevant DNA methylation markers were identified using a 5AZA-induced gene expression approach.
  
  
  CONCLUSIONS
  These breast cancer-relevant, FNA-optimized DNA methylation markers may have value as surrogate endpoint biomarkers in RP-FNA studies.
authors.affiliation: Authors' Affiliations: Departments of Surgery, Pathology, Clinical Sciences, and Pathology, Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center; and Department of Molecular and Cell Biology, Center for Systems Biology, The University of Texas, Dallas, Texas.
authors.forename: Dawei
authors.initials: D
authors.lastname: Bu
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Lewis
authors.forename: Venetia
authors.initials: V
authors.lastname: Sarode
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.forename: Xiaotu
authors.initials: X
authors.lastname: Ma
authors.forename: Aaron M
authors.initials: AM
authors.lastname: Lazorwitz
authors.forename: Roshni
authors.initials: R
authors.lastname: Rao
authors.forename: Marilyn
authors.initials: M
authors.lastname: Leitch
authors.forename: Amy
authors.initials: A
authors.lastname: Moldrem
authors.forename: Valerie
authors.initials: V
authors.lastname: Andrews
authors.forename: Adi
authors.initials: A
authors.lastname: Gazdar
authors.forename: David
authors.initials: D
authors.lastname: Euhus
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D044963:Biopsy, Fine-Needle; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004273:DNA, Neoplasm / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006801:Humans; D008875:Middle Aged
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm